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Environment International
journal homepage:www.elsevier.com/locate/envintHazardous e
ffects of silver nanoparticles for primary producers in
transitional water systems: The case of the seaweed Ulva rigida C. Agardh
Andrea Augusto Sfriso
a,⁎, Michele Mistri
a, Cristina Munari
a, Isabella Moro
b,
Mohammad Wahsha
c, Adriano Sfriso
d, Abdul-Salam Juhmani
daDepartment of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara 17, 44121 Ferrara, Italy bDepartment of Biology, University of Padova, Via U. Bassi, 58/B, 35131 Padova, Italy
cMarine Science Station, The University of Jordan, Aqaba branch, Jordan
dDepartment of Environmental Sciences Informatics and Statistics, Ca’ Foscari University of Venice, Via Torino 155, 30170 Mestre, Italy
A R T I C L E I N F O
Handling Editor: Frederic Coulon
A B S T R A C T
The acute toxicity of citrate capped silver nanoparticles (AgNP) and silver nitrate was evaluated on the marine macroalga Ulva rigida C. Agardh (1823). Silver bioaccumulation, ultrastructural chloroplast damages verified by TEM microscopy, inhibition of primary production, neutral lipid production and oxidative stress were observed after 24 h of exposure to AgNP. The toxic effects of silver nitrate in artificial seawater started from a con-centration of 0.05 ppm and was more toxic than AgNP that produced effects from a concentration of 0.1 ppm. However only AgNP induced lipid peroxidation in U. rigida. The addition of natural organic and inorganic ligands, represented by transparent exopolymer particles (TEP) and clay, drastically reduced AgNP acute toxicity in a ratio AgNP:ligand of 1:100 and 1:200, respectively. Thefindings suggest a marked toxicity of Ag on marine macroalgae which however should be mitigated by the high natural ligand concentrations of the transitional environments.
1. Introduction
The development of nanotechnology led to a rapid expansion of nanomaterials that are likely to become a source of many different nanoparticles (NP) discharged into the environment (Gambardella et al., 2015). Different unique properties of NP like high specific surface area and mobility, could potentially lead to unexpected health or en-vironmental hazards (Maynard et al., 2006). The use of silver colloids and nanoparticles have become a topic of interest in many biotechno-logical applications, attributed to their consolidated antimicrobial and biocidal properties (Fabrega et al., 2011;Rai et al., 2009). A survey on the patent search engine“Espacenet” (European Patent Office, 2019) for“silver nano” reveals an exponential trend in the release of > 5000 patents from the year 2000 and the 52% of these were recorded in the last 5 years. The same exponential trend can be found in the increasing scientific interest on silver nanoparticles documented in peer review journal publications from the early 2000s (McGillicuddy et al., 2017). Various applications of AgNP in many domestic and industrial products include: healthcare and cosmetics, clothing, food packaging,
paints, laundry additives, home appliances and medical devices(Zhang et al., 2015). Moreover 53% of the EPA (Environmnetal Protection Agency)-registered biocidal silver products are reported to contain AgNP (Nowack et al., 2011). The steady increase in consumer products released to the market, containing silver nano-compounds, could reflect in an increasing dispersion and diffusion of silver into the environment. AgNP can be discharged during synthesis, manufacturing and use of these products as well as the recycling and disposal processes (Köhler et al., 2008).
Little systemic toxicity was reported in vivo toward humans for the doses reported to be toxic for microbes and parasites (Hadrup and Lam, 2014;Le Ouay and Stellacci, 2015;Stoddard et al., 2013;van der Zande et al., 2012). This probably contributed to the widespread use of AgNP and at the same time increased the concern of researchers for thefinal receiving environments. In fact, Ag is considered the second most toxic metal (after Hg) for aquatic organisms (Kennedy et al., 2010).
The possible adverse effects of increased environmental exposure to AgNP include the development of silver-resistant bacteria (Gupta et al., 1999; Percival et al., 2005; Silver, 2003) and the impairment and
https://doi.org/10.1016/j.envint.2019.104942
Received 11 December 2018; Received in revised form 15 June 2019; Accepted 17 June 2019
Abbreviations: AgNP, silver nanoparticles; CTRL, control; LPO, lipid peroxidation; MDA, malondialdehyde; NP, nanoparticles; PP, primary production; TEP, transparent exopolymer particles; TEM, transmission electron microscopy; TWS, transitional water systems
⁎Corresponding author.
E-mail address:asfriso@hotmail.it(A.A. Sfriso).
0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
deterioration of aquatic organisms and environments, especially pri-mary producers (Asharani et al., 2008;Choi et al., 2008;Griffitt et al., 2008; Navarro et al., 2008). AgNP toxicity mechanisms include: membrane adhesion and disruption; alterations in proton-pump func-tion; lipopolysaccharide degradafunc-tion; enzymes inactivafunc-tion; cellular phosphate management disruption; inhibition of DNA synthesis, ribo-some denaturation and ROS production (Moreno-Garrido et al., 2015). To date, the vast majority of research involving Ag and AgNP toxicity on primary producers has been focused mainly on freshwater species and microalgae with little concern toward both marine species and macroalgae (Moreno-Garrido et al., 2015;Navarro et al., 2008). Mac-roalgae in transitional water systems (TWS) are important marine pri-mary producers, directly affecting the following levels of the trophic chain and consequently the final quality state of many coastal en-vironments (Sfriso et al., 2009, 2014).
The present study aims to investigate the interactions of AgNP and AgNO3with the most common primary producer of TWS, the seaweed
Ulva rigida C. Agardh (1823) utilizing biochemical and physiological biomarkers. Moreover, we investigate the effects of TWS natural or-ganic and inoror-ganic ligands on the toxicity of AgNP.
2. Material and methods 2.1. Experimental design
We evaluated the effects of silver in the form of citrate capped AgNP on a model primary producer organism of TWS: the macroalga U. rigida, a biological indicator species for metal bioaccumulation (Favero et al., 1996). For comparison the effects of AgNO3as source of Ag+was also
tested in parallel in order to identify AgNP specific toxic behaviors. The reactions of U. rigida were examined by lipid peroxidation (LPO), pri-mary production (PP), ultrastructural cellular alterations (by TEM imaging) and epifluorescence microscopy. A second experiment was performed to evaluate the effects of AgNP on U. rigida, examining PP and LPO, in presence of two common natural compounds of TWS: transparent exopolymer particles (TEP) and clays.
2.2. Reference study area
The Venetian Lagoon (Italy) was selected in this study as reference site to simulate the TWS conditions for the toxicity test. This lagoon is influenced by Venice town, where there is no sewer system and drains convey waters to canals. In this lagoon, metal pollution was found in sediments mainly due to the pollution released in the past by the Porto Marghera industrial area and the town discharges (Frignani et al., 1997; Masiol et al., 2014). Two rivers bring freshwater near the town from the mainland (Osellino and Dese rivers) in areas with high water residence time that can reach up to 60 days (Guerzoni and Tagliapietra, 2006). 2.3. Nanoparticles synthesis and characterization
AgNP were synthesized with the citrate reducing method reported inRatyakshi and Chauhan (2009). The plasmon resonance peak max-imum was measured with an UV-VIS spectrophotometer (Jenway, Germany). The size distribution of the nanoparticles was characterized through a transmission electron microscope (TEM) FEI Tecnai G2 (Hillsboro, Oregon) and three images of AgNP from TEM were pro-cessed by ImageJ software for the evaluation of particle size by Feret diameter.
2.4. Tested seaweeds and experimental setup
Artificial seawater (ASW) was prepared dissolving in one litter of Milli-Q water: NaCl: 24.6 g, KCl: 0.67 g, CaCl2:1.36 g, MgSO4x7H2O:
6.29 g, MgCl: 4.66 g, HNaCO3: 0.18 g, according toKester et al. (1967).
All the glassware was cleaned with “Contrad” detergent, soaked
overnight in HNO3 1%, to remove any trace metal elements, and
eventually rinsed with milli-Q water and HNaCO31% buffer solution.
Small (young) thalli of the seaweed U. rigida (max. 5 cm long) were collected in spring from the artificial rocky shores of the Lagoon of Venice in an area with high water renewal of the Lido island. The seaweed thalli were kept in seawater for 3 hours (h) for acclimation until their employment in the experimental tests.
The effects of both AgNO3and AgNP were tested in artificial
sea-water in concentration ranges from 0.01 to 120 ppm for LPO and from 0.1 to 20 ppm for PP. The toxicity on U. rigida was examined after 24 h by PP and LPO analyses, employing for each tested concentration, three flasks (100 mL) with 500 mg of fresh seaweed, equivalent to an infield standing crop of 1 kg m−2. The experiments were carried out at 22 °C. 2.5. Lipid peroxidation
Levels of MDA in U. rigida were measured according to Wahsha et al. (2012). A 100-mg fresh macrophyte sample was homogenized in 5 mL solution of 0.25% thiobarbituric acid in 10% trichloroacetic acid, and incubated at 95 °C for 30 min, followed by quick cooling and cen-trifuged at 10,000g for 10 min. The absorbance of the clear supernatant was read spectrophotometrically at 532 nm and correction for un-specific turbidity was done by subtracting the absorbance of the sample at 600 nm. The LPO was expressed as malondialdehyde (MDA) milli-mols formed per gram of fresh tissue (mmol g−1fw), using a molar extinction coefficient of 155 mM−1cm−1. Experiments were performed
in triplicate.
2.6. Primary production
Primary production was measured as changes in dissolved oxygen (DO) concentration at 22 °C after 24 h of AgNP/AgNO3 exposure
(Odum, 1956) by an oximeter (model Oxi 196, Wissenschaftlich–-Technische Werkstatten GmbH), equipped with a battery stirrer BR190. Two sets of three replicates for each concentration were tested: one set was kept in dark, where respiration depleted oxygen. The other set was kept under continuous light of 30μmol (red and blue LED ratio 2:1), where photosynthesis produced oxygen. The sum of photosynthesis and respiration returns the primary production score, which was expressed as mg of elemental oxygen per gram of dry tissue produced in 1 h during the process (mg O2g−1dw−1h−1).
2.7. Transmission electron microscopy (TEM)
To investigate the ultrastructural alterations of U. rigida induced by AgNP and AgNO3, control and treated thalli werefixed in 3%
glutar-aldehyde in 0.1 M cacodylate buffer (pH 6.9) for 2 h at 4 °C. After fixation, thalli were treated according toMoro et al. (2002). Samples embedded in an Epon-Durcupan ACM mixture were cut through a Reichert Ultracut S ultramicrotome and the ultrathin sections were poststained with lead citrate and examined with a transmission electron microscope (TEM - FEI Tecnai G2) operating at 100 kV.
2.8. Epifluorescence microscopy
Nile red lipid staining protocol reported inStorms et al. (2014)was applied to the samples treated with different AgNP and AgNO3
con-centrations. Chlorophyll-a autofluorescence and lipid fluorescence were visualized byfluorescent microscope (Olympus BX51 Microscope) and images were processed by ImageJ software.
2.9. Metal analysis
U. rigida thalli were rinsed with artificial seawater, dried with a tissue and maintained in oven at 50 °C for 48 h. The samples were homogenized and 100 mg were digested in acid-treated Teflon vessels
by 3 mL of HNO3, 3 mL of HClO4and 4 mL of Milli-Q water for 2 h at
130 °C (Rigollet et al., 2004). The volume of the extracts was brought to 50 mL, centrifuged at 5000 rpm for 20 min, transferred in polyethylene containers and maintained at 4 °C until analysis. Silver analysis was carried out by Perkin Elmer ICP-OES 5300DV. All the analyses were done in triplicate.
2.10. TEP and turbidity effect
The effect of TEP and turbidity on the toxicity of AgNP was eval-uated using two concentrations of AgNP (0.5 and 5 ppm) by addition of sulphated agar (anionic polysaccharide) and clay. The TEP were syn-thesized by sulphated agar purified according to Sfriso et al. (2017) from Gracilariopsis longissima (S.G. Gmelin) Steentoft, L.M. Irvine et Farnham then dissolved in boiling water ad poured in cold ASW. Pharma-grade purified clay was purchased from the market and tested in the range from 10 to 100 ppm.
2.11. Statistical analysis
All treatments were done in triplicates. Means and standard devia-tions were calculated for each treatment. The Shapiro-Wilk test was used to check for normality and significant differences between control samples and AgNP/AgNO3exposed algal samples were determined by
Mann-Whitney U test where p-value of 0.05 or less was considered to be significant. All the analyses were performed by using Statistica version 10.1 (Statsoft, Inc. Tulsa, USA).
3. Results
3.1. Nanoparticle characterization
The citrate capped AgNP displayed a surface plasmon resonance peak within the range from 412 to 416 nm. The AgNP mean size and distribution, determined by ImageJ particle analysis on TEM micro-scopic images, showed a mean Feret diameter of 35 ± 9.1 nm (Fig. 1a). The particle size distribution, reported in histogram (Fig. 1b), showed that > 90% of the AgNP ranged from 20 to 50 nm in size. Results about behavior and fate of AgNP and AgNO3in ASW were included in
Sup-plementary material. 3.2. Silver uptake and toxicity
Silver nanoparticles were bioaccumulated in U. rigida tissues from the solution. The metal analysis showed that the uptake of silver follows the Langmuir isotherm equation (Fig. 2) with a saturation point in U.
rigida of 11.3μg Ag g−1fw−1from a 5 ppm AgNP solution. A relatively
high accumulation was found in U. rigida already from 1 ppm AgNP solution reaching 8.50μg Ag g−1fw−1in the seaweeds.
The MDA levels are presented inFig. 3a, in agreement with the bioaccumulation results, displayed for AgNP, the lowest observed ad-verse effect level was at 1 ppm, the first concentration significantly different from control (Test U Mann-Whitney: d.f. 9, p = 0.01).
The oxidative stress generated by AgNP steadily increased up to concentrations of 30 ppm AgNP, then started decreasing in conjunction with cell death, as ascertained by light microscope (data not shown). For comparison, the oxidative stress generated by AgNO3was also
in-vestigated from 0.01 to 120 ppm, but the MDA values did not diverge from the control. However, the AgNO3produced a toxic effect, reducing
the PP by 68%, starting from the concentration of 0.05 ppm (Fig. 3b). Negative values of PP were found from 0.1 to 5 ppm, corresponding to consumption of oxygen due to respiration to cope with the toxic effect of silver. This also highlighted that respiration processes were still working up to 1 ppm. AgNP generated instead an eutrophication effect from 0.01 to 0.07 ppm. The adverse effects manifested only from 0.1 ppm with the complete inhibition of PP at 5 ppm AgNO3in
con-junction with the maximum bioaccumulation of silver in the seaweed. The oxidative stress measured by MDA started for AgNP after primary production was inhibited but this increase in MDA was found only for AgNP and not for AgNO3. The AgNO3at 5 ppm induced cell lysis and
death. Conversely, AgNP completely inhibited PP but did not induce lysis, as verified by epifluorescence, and produced a steady increase of oxidative stress.
Fig. 1. a: TEM image of AgNP.
b: size distribution of AgNP. Data are mean ± s.d. (n = 3).
Fig. 2. Silver bioaccumulation in U. rigida from AgNP solutions. Data are mean ± s.d. (n = 3).
3.3. Epifluorescence imaging
The microscope investigation results comply with the effects of AgNP and AgNO3on PP. The control (CTRL) images displayed healthy
cells and the respective red auto-fluorescence of chlorophyll-a (Fig. 4a
and b). At 0.05 ppm of AgNP no significant effects were noticed and only chlorophyll-a auto-fluorescence was observed (Fig. 4c). Con-versely, for the same concentration of AgNO3 (Fig. 4d) small yellow
dots appeared inside the cells due to Nile-red binding neutral lipids. At 0.5 ppm both AgNP (Fig. 4g and e) and AgNO3(Fig. 4f) displayed an Fig. 3. a: MDA levels in U. rigida at different concentrations of AgNP (•) and AgNO3(□). Data are mean ± s.d. (n ≥ 3).
b: PP in U. rigida at different concentrations of AgNP (•) and AgNO3(□). Data are mean ± s.d. (n ≥ 3).
Fig. 4. Light and epifluorescence microscope (400×) images of samples treated with AgNP and AgNO3(0.05, 0.5, 5 ppm) in comparison with control without toxicants (CTRL).
increased number of stained cells, which density seemed higher for AgNP solutions. Eventually, at 5 ppm all the cells were stained (Fig. 4g), but for AgNO3the effect was more marked with cell disruption, lysis
(Fig. 4h) and lipid dispersion, creating a misty effect onto the algal tissues.
3.4. Ultrastructural analysis
Transmission electron microscope analysis carried out to investigate the ultrastructural modifications of U. rigida induced by AgNP and AgNO3, confirmed the results obtained at light microscope. CTRL
images showed cells with a chloroplast characterized by regular thy-lakoids and with an evident pyrenoid surrounded by starch (Fig. 5a and b). At 0.05 ppm of AgNP no relevant modifications were noticed (Fig. 5c), but at 0.05 ppm of AgNO3thylakoidal membranes appeared
swollen (Fig. 5d). At 0.5 ppm of both AgNP (Fig. 5e) and AgNO3
(Fig. 5f) some ultrastructural changes were evident. In particular, in the AgNP treated thalli, cells showed chloroplasts with stacked thylakoids, complete pyrenoids and some small electron-dense inclusion bodies, probably neutral lipids, as detected by Nile-red staining at the epi-fluorescence microscope (Fig. 5e). Instead, in the AgNO3, chloroplasts
exhibited swollen thylakoids and some osmiophilic granules (Fig. 5f).
At AgNP 5 ppm the cells showed chloroplasts characterized by a large number of osmiophilic granules, remaining pyrenoids and swollen thylakoids (Fig. 5g). This was more evident at AgNO3 5 ppm, where
more damaged chloroplasts showed disorganized thylakoidal mem-branes with many osmiophilic globules surrounding a residual of pyr-enoid (Fig. 5h). Moreover, TEM analysis did not highlight a bioaccu-mulation of silver nanoparticles inside the cells of U. rigida. There was, in fact, a very impenetrable cell wall (Fig. S1), besides to an external exopolymeric substance layer (Fig. S2), that constituted a barrier, preventing the access of larger particles, and consequently a direct in-ternalization of Ag nanoparticles.
3.5. TEP and turbidity effect
The effect of TEP addition on AgNP toxicity was evaluated by MDA and PP. MDA values verged toward the control with increasing TEP concentrations (Fig. 6a and b). The tests showed a 50% reduction of the effects of 0.5 ppm AgNP with the addition of 10 ppm of TEP and a complete detoxification at a TEP concentration of 50 ppm. The same TEP concentrations employed in the test did not produce any significant variations on toxicity of 5 ppm AgNP. Eventually TEP produced de-toxification in a ratio AgNP:TEP of 1:100.
Fig. 5. Transmission electron microscope images of samples treated with AgNP and AgNO3(0.05, 0.5, and 5 ppm) in comparison with control without toxicants (CTRL). (c = chloroplast; t = thylakoid; p = pyrenoid; cw = cell wall; lb. = lipid body).
A similar behavior was found for the effect of clay on AgNP toxicity, evaluated by MDA (Fig. 7a). The values displayed only a small reduc-tion of the adverse effects due to clay addition to the solutions. PP values displayed instead an almost complete detoxification of 0.5 ppm AgNP by 100 ppm of clay with an AgNP:clay ratio of 1:200. Conversely, the toxic effects of a 5 ppm AgNP solution could not be reduced even by 100 ppm of clay in solution (Fig. 7b). Eventually both clay and TEP at high concentration greatly reduced AgNP toxicity, in terms of PP in-hibition, but only TEP also reduced the oxidative stress.
4. Discussion
Despite the high ecological and economical importance and high productivity of TWS, they represent a sink for many pollutants in-cluding metals (Guerzoni and Tagliapietra, 2006; Ridgway and Shimmield, 2002). However, until recently, little attention was paid to silver contamination despite the increased level of production and re-lease of AgNP based products. Different marine organisms were ex-amined for AgNP toxicity, but only few articles discuss the effects of silver and AgNP on marine macroalgae, which are the dominant pri-mary producers in TWS (Moreno-Garrido et al., 2015).
The results of this study confirm the high phytotoxicity of silver from both AgNP and AgNO3 on the marine macroalga U. rigida as
previously reported for microalgae (He et al., 2012). The toxicity of silver from AgNO3 was tenfold higher in comparison with AgNP
(Boenigk et al., 2014) moreover AgNO3produced a severe inhibition of
primary production in U. rigida starting from 0.05 ppm. This result was in accordance with the previously reported value of 0.045 ppm, the lowest available silver concentration at which a toxic response was measured in Ulva (Turner et al., 2012). At this concentration AgNP did not produce any PP impairment, but an eutrophication effect was
measured up to 0.070 ppm and PP inhibition due to AgNP was found at higher concentrations (0.100 ppm).
The toxic effect of silver from AgNO3was higher than that from
AgNP as confirmed by the epifluorescence microscopic examination. The increase of neutral lipids, highlighted by the nile red staining, was associated with membrane destabilization by metal pollution (Kumar et al., 2010). The destabilization of the thylakoidal membrane system was confirmed by observations at the transmission electron microscope, where chloroplasts, of both AgNP and AgNO3treated cells, appeared
strongly damaged, with reduced thylakoidal membranes and evident dilatations, besides a large number of osmiophilic globules. These morphological changes usually are correlated with functional altera-tions in the chloroplasts, measured by photosystem I and II activities (Hall et al., 1972).
Negative PP values were found for U. rigida, caused by the re-spiration process in the absence of photosynthetic activity, for AgNO3
concentrations between 0.1 and 1 ppm. This suggests that mitochon-drial activity was still acting at AgNO3concentrations up to 1 ppm,
while the chloroplast photosystem in the range 0.045–0.075 ppm was already inhibited (Turner et al., 2012). Complete inhibition of both photosynthesis and respiration activities occurred at 5 ppm as found by Teodoro et al. (2011), who reported impairment of mitochondrial ac-tivity between 2 and 5 ppm. Eventually, both AgNP and AgNO3 at
5 ppm produced the complete PP inhibition, but only AgNP produced an oxidative stress. Conversely, AgNO3induced cell death and lysis but
no oxidative stress measurable by MDA. These differences are probably due to the routes by which toxicity is carried out. Copper transporter proteins were reported to mediate the transport of Ag+into the cells (Blaby-Haas and Merchant, 2012) and could be an important route of silver uptake and toxicity. In regard to AgNP we have no evidence of direct internalization of the particles. Many other studies report no Fig. 6. a: MDA concentration in U. rigida for 5 ppm (□) and 0.5 ppm AgNP (•) at different concentrations of TEP. Data are mean ± s.d. (n = 3).
b: PP in U. rigida for 5 ppm (□) and 0.5 ppm AgNP (•) at different concentrations of TEP. Data are mean ± s.d. (n = 3).
Fig. 7. a: MDA concentration in U. rigida for 5 ppm (□) and 0.5 ppm AgNP (•) at different clay concentrations. Data are mean ± s.d. (n = 3) b: PP in U. rigida for 5 ppm (□) and 0.5 ppm AgNP (•) at different clay concentrations. Data are mean ± s.d. (n = 3).
evidence of AgNP direct uptake (Zheng et al., 2019), though we proved that silver was bioaccumulated reaching at 24 h the saturation point from an AgNP solution of 5 ppm. The same concentration induced a complete PP inhibition both in AgNO3and AgNP solutions, highlighting
that silver definitely produced toxic effects inside the cell. AgNP was reported to accumulate mainly on the cell surface (Hu et al., 2018) with little internalization in microalgae (Zheng et al., 2019). The pore size of the cell wall mat in algae was estimated to be of about 7-20 nm, ex-cluding the access of larger particles (like the ones tested in this study) to the cell surface (Navarro et al., 2008; Zemke-White et al., 2000). However, the measure of oxidative stress in U. rigida reveals membrane destabilization and reactive oxygen species production. The oxidative stress effect was not produced by Ag+from AgNO
3but only by the
AgNP and could be related to the effect of Ag bulk forms or to AgNP oxidation and dissolution with the production of both Ag+and free radicals in proximity of the cell surface (Batchelor-mcauley et al., 2014; Oukarroum et al., 2012;Zewde et al., 2016). The free Ag+released in
proximity of the cell wall could be complexed by citrate (available at a concentration 10 times higher than Ag in the AgNP solution) or by the abundant Cl−in ASW. This would create soluble AgClx(1-x)complexes
(Batchelor-mcauley et al., 2014; Levard et al., 2012) able to pass through the cell wall mat, reaching the membrane where Ag+can be
internalized. These hypotheses are supported by the fact that no par-ticles were observed inside the cell wall in the TEM micrographs at larger magnifications; moreover, a barrier effect due to exopolimeric substances produced by algae was reported hindering the AgNP inter-nalization (Zheng et al., 2019).
On the large scale the behavior and fate of AgNP in the aquatic environments are the result of the complex interactions between the chemistry of nanoparticles and the physical, chemical and ecological properties of the receiving environments (Le Ouay and Stellacci, 2015). Some investigations reported the role of different kinds of natural or-ganic ligands, such as the adsorption of dissolved oror-ganic substances to the surface of AgNP, changing their superficial properties (e.g. redox potential, charge, coating) and Ag+release (El Badawy et al., 2010; Levard et al., 2012;Liu and Hurt, 2010;Xiu et al., 2011). The role of dissolved organic carbon/matter was reported to alter dissolution, colloidal stability and toxicity of citrate-capped AgNP (Pokhrel et al., 2013). Conversely, few data are available on the effects of natural particulate matter, both organic (TEP) and inorganic (clay).
In the Lagoon of Venice, average values of particulate suspended matter of 40 ppm were reported with peaks of 115 ppm (Sfriso and Sfriso, 2017). Moreover, the northern Adriatic Sea is also characterized by high TEP concentrations, ranging from 2 to 44 ppm with peak values over 400 ppm (Engel, 2001;Passow, 2002).
We observed a distinct effect, of both TEP and clay (with average concentrations of the Venice Lagoon) on the detoxification of AgNP up to 0.5 ppm. A similar detoxification of AgNP due to external exopoly-meric substances was reported (Miao et al., 2009;Zheng et al., 2019) and anionic polysaccharides such as k-carrageenan and xanthan gum (Elsupikhe et al., 2015;Xu et al., 2014) were used as capping agents for AgNP due to their affinity for the particles. Concurrently,Dong et al. (2009)used the clay for AgNP immobilization during synthesis ascer-taining the clay binding action on the particles. Although both clay and TEP greatly reduced AgNP toxicity as PP only TEP reduced the oxida-tive stress. This could be explained by the antioxidant properties of the sulphated polysaccharides (Francavilla et al., 2013;Souza et al., 2012), that could reduce the reactive oxygen species produced by AgNP dis-solution.
Some hypothesis and models on AgNP release in the environment were proposed and different amounts of AgNP into the aquatic eco-systems were predicted and measured in the part per trillion range. Gottschalk et al. (2009)model predicted silver concentration ranges for surface waters and sewage treatment plant effluents of 0.07–0.11 ppt and 21–42 ppt, respectively. Moreover, predicted environmental con-centrations of AgNP in water compartment were estimated in the range
of 30 to 80 ppt (Mueller and Nowack, 2008). Similar values, in the ppt range, were measured byLiang-Saw et al. (2002)in Colorado surface waters and sewage effluents (1.8–537 ppt). Eventually, for a risk esti-mationBlaser et al. (2008)took into account silver concentration va-lues in the range from 40 to 320 ppt from sewage effluent. The esti-mated concentrations for freshwater in the parts per trillion range were lower than the values hazardous for seaweeds and aquatic organisms. The NP released into the environment are expected to display brief residence times in the water column and are expected to aggregate and accumulate in sediments and benthic communities, especially when salinity increases near the sea decreasing the stabilizing effect of the dissolved organic matter on AgNP (Burgess et al., 2014). Indeed, Chinnapongse et al. (2011) reported agglomeration of 80% of the particles within 1 h and the complete disappearance after 10 h in ac-cordance with our results. However, the results we provided (Supple-mentary material) also highlighted the transformation of AgNP to AgCl in ASW eventually dissolving in 24 h for ca. 60–70% probably leading to the formation of the highly mobile forms AgClx(1-x)complexes. In this
context, estuaries and lagoons with high water residence time (> 15–30 days) could accumulate silver that may generate toxic effects in the long term.
It is unlikely that concentrations lower than 0.5 ppm could pose a threat to primary producers in TWS, where the combined effect of both TEP and turbidity (usually high in these environments) should neu-tralize the adverse effects on primary producers. The few data found in literature on silver environmental contamination report very low con-centrations (in the ppt range) in the water column, under the values found to be toxic for U. rigida, but the silver bioconcentration factors can range from 104 to slightly beyond 105 (Toni, 1999). Moreover,
silver concentration values from 3.5 to 10.3μg g−1dw were reported for sediments and mussels in the Venice Lagoon (Giusti and Zhang, 2002), warning about the presence of this element, not so negligible. Eventually data on long-term exposition to low levels of silver and AgNP is still missing and the sequestration of NP by TEP and clays could extend the lifespan of NP into the environment.
5. Conclusions
The obtained results from different lines of evidence converge in describing the acute toxicity of AgNP and AgNO3in U. rigida. Silver
bioaccumulation, altered chloroplast, inhibition of primary production and lipid peroxidation were assessed in the seaweed after 24 h of ex-posure. The natural organic and inorganic ligands present in transi-tional environments were also found to detoxify the AgNP at con-centrations up to 0.5 ppm. AgNP concon-centrations under this value do not seem to pose a threat for primary producers in transitional waters. However, further long-term investigations are recommended to ex-amine the response of seaweeds at very low AgNP concentrations (ppb/ ppt) that can be bioaccumulated in confined water bodies with high water residence time.
Acknowledgements
The funds are provided by Ca'Foscari University. Appendix A. Supplementary data
Supplementary data to this article can be found online athttps:// doi.org/10.1016/j.envint.2019.104942.
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